Endophytic fungi from Pteromischum sp. plant, compounds and methods of use

ABSTRACT

The present disclosure relates to endophytic fungi from higher plants such as a  Pteromischum  sp. plant, and to extracts and compounds from such fungi that have desirable biological activities, such as antifungal and immunosuppressive activities. The present disclosure further relates to compositions comprising such extracts and compounds, as well as methods of making and using the compositions.

CROSS REFERENCE TO RELATED APPLICATION

This application is a divisional of U.S. application Ser. No.12/741,804, filed May 6, 2010, now U.S. Pat. No. 8,080,256 which is a371 national phase entry of PCT/US2008082678, filed Nov. 11, 2008 andclaims the benefit of U.S. Provisional Patent Application No.60/986,946, filed Nov. 9, 2007, all of which are herein incorporated byreference in their entirety.

FIELD OF THE DISCLOSURE

The present disclosure relates to endophytic fungi from higher plantssuch as a Pteromischum sp. plant, and to extracts and compounds fromsuch fungi that have desirable biological activities, such as antifungaland immunosuppressive activities. The present disclosure further relatesto compositions comprising such extracts and compounds, as well asmethods of making and using the compositions.

BACKGROUND OF THE DISCLOSURE

Endophytes, microorganisms that reside in the tissues of living plants,are relatively unstudied and potential sources of novel natural productsfor exploitation in medicine, agriculture and industry. It is worthy tonote, that of the nearly 300,000 plant species that exist on the earth,each individual plant is host to one or more endophytes. Only a handfulof these plants have ever been completely studied relative to theirendophytic biology. Consequently, the opportunity is great to find newand interesting endophytic microorganisms among myriads of plants indifferent settings, and ecosystems.

Currently, endophytes are viewed as an outstanding source of bioactivenatural products because there are so many of them occupying literallymillions of unique biological niches (higher plants) growing in so manyunusual environments. While the symptomless nature of the occupation ofplant tissues by endophytes has prompted focus on symbiotic ormutualistic relationships between endophytes and their hosts, theobserved biodiversity of endophytes suggests they can also be aggressivesaprophytes or opportunistic pathogens. Both fungi and bacteria are themost common microbes existing as endophytes (Bacon and White, MicrobialEndophytes. Marcel Dekker Inc., N.Y., 2000). For example, some of theseorganisms make compounds now exploitable as anticancer drugs,antibiotics, and antioxidants.

There is a need for more and better antimycotics, as the humanpopulation is developing more fungal infections. This is particularly anissue with immunosuppressed patients, such as HIV/AIDS patients,patients with organ-transplants, and anyone who must takeimmunosuppressive drugs. In both cases, patients with these difficultieshave immune systems that are weakened. Antifungal agents that arecurrently available, such as cyclosporin A, are toxic to the subject,and often ineffective against the fungal pathogen.

Since the discovery of cyclosporin A from Trichoderma polysporum in1976, it has been the principal immunosuppressive agent used in medicine(Ruegger et al., Hel. Chim. Acta. 59: 1075-1092, 1976). Presently,cyclosporin A, along with tacrolimus (FK506) and sirolimus (rapamycin)are three immunosuppressants which act on CD4⁺ T cells used in clinicalpractice. These compounds have gained wide spread acceptance for use inorgan and tissue transplantation, various autoimmune diseases and withsome other non-autoimmune inflammatory diseases. However, all threedrugs can cause nephrotoxicity (Daoud et al., Epilepsia 48: 834-836,2007). In addition, cyclosporin A and tacrolimus can cause neurotoxicityand beta-cell toxicity (Tanabe, Drugs 63: 1535-48, 2003; Froud et al.,Cell Transplant 15: 613-620, 2006). Cyclosporin A can cause more seriousnephrotoxicity, hypertension and hyperlipidaemia in comparison totacrolimus (Andoh et al., Kidney Int. 50: 1110-1117, 1996). Novelcompounds with low toxicity that act in an effective and useful mannerwill contribute to the arsenal of substances that act to suppress theimmune system and will be especially helpful to those with autoimmunediseases and organ recipients.

There is also a need for environmentally sound ways to control pests andpathogens (Overton, Ecologically Based Pest Management—New Solutions fora New Century. Natl. Aca. Press. Washington D.C., 1996). In the past,the major source of pesticidal agents came from organic synthesis.Recently, interest has increased for using more environmentally friendlymethods in agricultural production, including naturally-occurringbiological compounds.

SUMMARY

Provided herein is an endophytic Colletotrichum (previously known asVolutella) sp., associated with a Pteromischum sp. plant growing in atropical forest in Costa Rica, which is capable of producing antifungalor immunosuppressant compounds. In an example, a specific Colletotrichumsp. isolate, referred to as isolate C-12, is disclosed that possessesantifungal and immunosuppressant activities. For example, the disclosedisolate C-12 was capable of inhibiting pathogenic fungi includingBotrytis cinerea, Sclerotinia sclerotiorum, or Rhizoctonia solani.

The present disclosure also relates to extracts, compositions andcompounds generated from endophytic Colletotrichum sp. isolates, such asisolate C-12, including specifically, extracts, compositions andcompounds that have immunosuppressive or antimycotic activity. Forexample, exemplary compounds can include cyclic lipopetides withantimycotic and immunosuppressive activities.

The present disclosure further relates to methods for producing abiological agent, including an endophytic Colletotrichum sp.; an extractof an endophytic Colletotrichum sp.; or a compound obtained from theendophyte (e.g., a compound or mixture of compounds having antimycotic,immunosuppressive, or other biological activity). In an example, themethod includes cultivating a strain of endophytic Colletotrichum sp.and recovering the biological agent from the culture medium or from anextract prepared from Colletotrichum cells.

Also provided are methods of suppressing an immune response in asubject, such as a subject with an autoimmune disease, non-autoimmuneinflammatory disease or in need of an organ or tissue transplant.

Additionally, methods of protecting plants against attack by a plantpathogen, such as fungi are provided. In an example, isolate C-12,including specifically extracts, compositions and compounds are used totreat fungal infections in plants.

The foregoing and other features will become more apparent from thefollowing detailed description, which proceeds with reference to theaccompanying figures.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a digital image of an exemplary environmental scanningelectron microscope (SEM) image of the sporodochial fruiting structureof Colletotrichum sp. isolate C-12 along with conidiospores and setaeprotruding from the structure (bar 20 μM). The inset shows a point driedimage (bar 50 μM).

FIG. 2 is a digital image of a proton nuclear magnetic resonance (NMR)spectrum of colutellin A taken in 100% deuterated methanol. The spectrumis consistent with that of a lipopeptide, with many methyl and methyleneresonances at 0.7-2.0 p.p.m. and other proton resonances on carbonsbearing nitrogen and oxygen further downfield.

FIG. 3 is a digital image of a mass spectrum of compounds taken byFourier transform ion cyclotron resonance (FTICR). Two clusters of ionswere detected, one protonated and the other sodiated. M1=1081.7,M2=1095.7, M3=1111.7, M4=1127.7 (M4 isotopic peak is well resolved inLC/MS). Since this spectrum represents the masses of the entire broadsilica gel column/HPLC peak, the M4⁺ Na⁺ peak is minimized.

FIG. 4 includes graphs illustrating that colutellin A (denoted by asquare) is less toxic to human peripheral blood mononuclear cells(PBMCs) than is cyclosporin A (denoted by an enclosed diamond). HumanPBMCs were treated with varying concentrations of colutellin A,cyclosporin A or equivalent amounts of DMSO (carrier), denoted bytriangle for 24 and 48 hours. The cells (PBMCs) were then stained with7-AAD and Annexin V directly conjugated to FITC or PE and subjected toflow cytometry. The percentage of necrotic (7-AAD+/Annexin V+),apoptotic (7-AAD-/Annexin V+) and viable (7-AAD-/Annexin V−) cells werecalculated using CellQuest software (BD Biosciences). Data isrepresentative of multiple studies. Error bars represent standarddeviations between triplicate treatment groups.

SEQUENCE LISTING

The nucleic acid and protein sequences listed in the accompanyingsequence listing are shown using standard letter abbreviations fornucleotide bases, as defined in 37 C.F.R. 1.822. Only one strand of eachnucleic acid sequence is shown, but the complementary strand isunderstood as included by any reference to the displayed strand.

SEQ ID NO: 1 is an amino acid sequence included with collutelin A.

SEQ ID NOS: 2 and 3 are oligonucleotide primer sequences used to detectthe ITS regions of C. dematium fungus.

DETAILED DESCRIPTION OF SEVERAL EMBODIMENTS I. Terms and AbbreviationsAbbreviations

AMU: Atomic Mass Unit

ESEM: Environmental Scanning Electron Microscope

FTICR: Fourier transform ion cyclotron resonance

HPLC: High Pressure Liquid Chromatography

LC/MS: Liquid Chromatography/Mass Spectrometry

MIC: Minimum Inhibitory Concentrations

NMR: Nuclear Magnetic Resonance

PBMC: Peripheral Blood Mononuclear Cell

SEM: Scanning Electron Microscope

UV: Ultraviolet

Terms

Unless otherwise noted, technical terms are used according toconventional usage. Definitions of common terms in molecular biology maybe found in Benjamin Lewin, Genes VII, published by Oxford UniversityPress, 2000 (ISBN 019879276X); Kendrew et al. (eds.), The Encyclopediaof Molecular Biology, published by Blackwell Publishers, 1994 (ISBN0632021829); and Robert A. Meyers (ed.), Molecular Biology andBiotechnology: a Comprehensive Desk Reference, published by Wiley, John& Sons, Inc., 1995 (ISBN 0471186341); and other similar references.

As used herein, the singular terms “a,” “an,” and “the” include pluralreferents unless context clearly indicates otherwise. Similarly, theword “or” is intended to include “and” unless the context clearlyindicates otherwise. Also, as used herein, the term “comprises” means“includes.” Hence “comprising A or B” means including A, B, or A and B.It is further to be understood that all amino acid sizes and molecularweight or molecular mass values given for the polypeptides areapproximate and are provided for description. Although methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the present disclosure, suitable methods andmaterials are described below. All publications, patent applications,patents, and other references mentioned herein are incorporated byreference in their entirety. In case of conflict, the presentspecification, including explanations of terms, will control. Inaddition, the materials, methods, and examples are illustrative only andnot intended to be limiting.

In order to facilitate review of the various embodiments of thisdisclosure, the following explanations of specific terms are provided:

Administer: To provide or give a subject an agent, such as colutellin A,by any effective route. Administration can be systemic or local.Exemplary routes of administration include, but are not limited to,oral, injection (such as subcutaneous, intramuscular, intradermal,intraperitoneal and intravenous), sublingual, rectal, transdermal (e.g.,topical), intranasal, vaginal and inhalation routes. For example, if thechosen route is intravenous, the composition is administered byintroducing the composition into a vein of the subject, and if thechosen route is intramuscular, the compositing is administered byintroducing the composition in to a muscle. In particular examples,agents (such as those including colutellin A) are administered to asubject having or at risk of developing a fungal infection, anautoimmune disease, a non-autoimmune inflammatory disorder or rejectingan organ or tissue transplant.

Agent: Any protein, nucleic acid molecule, compound, small molecule,organic compound, inorganic compound, or other molecule of interest.Agent can include a therapeutic agent, a diagnostic agent or apharmaceutical agent. A therapeutic or pharmaceutical agent is one thatalone or together with an additional agent induces the desired response(such as inducing a therapeutic or prophylactic effect when administeredto a subject). In an example, an agent includes an endophyticColletotrichum sp. isolate, such as isolate C-12. In a particularexample, an agent includes colutellin A.

Antimycotic: An agent that suppresses, inhibits, prevents or destroysthe growth of fungi.

Autoimmune disease: A disease in which the immune system produces animmune response (for example, a B cell or a T cell response) against anantigen that is part of the normal host (that is, an autoantigen), withconsequent injury to tissues. An autoantigen may be derived from a hostcell, or may be derived from a commensal organism such as themicro-organisms (known as commensal organisms) that normally colonizemucosal surfaces.

Exemplary autoimmune diseases affecting mammals include rheumatoidarthritis, juvenile oligoarthritis, collagen-induced arthritis,adjuvant-induced arthritis, Sjogren's syndrome, multiple sclerosis,experimental autoimmune encephalomyelitis, inflammatory bowel disease(e.g., Crohn's disease, ulcerative colitis), autoimmune gastric atrophy,pemphigus vulgaris, psoriasis, vitiligo, type 1 diabetes, non-obesediabetes, myasthenia gravis, Grave's disease, Hashimoto's thyroiditis,sclerosing cholangitis, sclerosing sialadenitis, systemic lupuserythematosis, autoimmune thrombocytopenia purpura, Goodpasture'ssyndrome, Addison's disease, systemic sclerosis, polymyositis,dermatomyositis, autoimmune hemolytic anemia, pernicious anemia, and thelike.

Biological Activity: An expression describing the effect of a substancesuch as an extract, composition or compound on living matter. In anexample, biological activity includes at least one of antifungal orimmunosuppressive activity. In one example, the extract, composition orcompound includes at least one specific endophytic Colletotrichum sp.isolate which has antifungal or immunosuppressive activity. For example,the extract, composition or compound includes at least colutellin A(molecular mass of 1127.7) which has antifungal and immunosuppressiveactivity.

Botrytis cinerea: A fungus that affects many plant species, includingwine grapes. In viticulture, it is commonly known as botrytis bunch rot;in horticulture, it is usually called grey mould or gray mold. Thefungus gives rise to two different kinds of infections on grapes. Thefirst, grey rot, is the result of consistently wet or humid conditions,and typically results in the loss of the affected bunches. The second,noble rot, occurs when drier conditions follow wetter, and can result indistinctive sweet dessert wines, such as Sauternes or the Aszú of Tokaj.

Colletotrichum denzatium: An endophytic fungus that has inhibitoryactivity to Botrytis cinerea. Colletrotrichum has the same fruitingstructure as Volutella. In the past, Colletotrichum sps., such as C.dematium, have been distinguished from Volutella by their pathogenicactivity whereas Volutella are non-pathogenic. Recently, non-pathogenicexamples of Colletotrichum sps. have been identified.

Described herein is a C. dematium fungus designated CR-12 (rDNA sequencefor CR-12 GenBank accession number EU3330193) that produces the novelpeptide antimycotic, colutellin A. Colutellin A is a cyclic lipopeptidewith a molecular mass of 1127.7, possesses bioactivities at leastagainst fungi (such as Botrytis cinerea and Sclerotinia sclerotiorum) aswell as immunosuppressive activities (including inhibiting theproduction of IL-2 from CD4). Colutellin A is the same compound that wasdisclosed in U.S. Patent Application No. 60/986,946 filed on Nov. 9,2007, but referred to therein as volutellin A. Based on initialtaxonomic studies, the compound was termed volutellin A because it wasbelieved to be produced from a Volutella sp. fungus. However, furthermolecular studies indicated that the fungus was C. dematium and not aVolutella sp. Therefore, the compound was renamed to reflect thisdistinction.

Cultivation: Intentional growth of an organism, such as an endophyticColletotrichum sp., in the presence of assimilable sources of carbon,nitrogen and mineral salts. In an example, such aerobic growth can takeplace in a solid or semi-solid nutritive medium, or in a liquid mediumin which the nutrients are dissolved or suspended. In a further example,the cultivation may take place on a surface or by submerged culture. Thenutritive medium can be composed of complex nutrients or can bechemically defined.

Disease: An abnormal condition of an organism that impairs bodilyfunctions.

Endophyte: Plant-associated microorganisms that live in the interstitialspaces of living plant tissues (Bacon & White, Microbial Endophytes.Marcel Deker Inc., N.Y., 2000). Higher plants may host one or moreendophytic microbes, which include fungi, bacteria, and actinomycetes.Endophytes reside in the tissues beneath the epidermal cell layers. Itis well understood that endophytic infections or associations areinconspicuous (Id.). As a result, the host tissues are transientlysymptomless and colonization of the tissues is internal to the surfaceof the plant. The exact physical relationship of the endophyte to theplant remains obscure, because it is extremely difficult, for example,by electron microscopic techniques, to find an endophyte within planttissues. The relationship that any given endophyte establishes with theplant likely varies from truly symbiotic to something bordering onpathogenic. In an example, a specific endophyte such as an endophyticColletotrichum sp. is isolated from one or more plants in which theendophytic Colletotrichum sp. produces antimycotic, immunosuppressiveactivities or other biologically active compounds of interest.

Immune response: A response of a cell of the immune system, such as aB-cell, T-cell, macrophage or polymorphonucleocyte, to a stimulus suchas an antigen. An immune response can include any cell of the bodyinvolved in a host defense response for example, an epithelial cell thatsecretes an interferon or a cytokine. An immune response includes, butis not limited to, an innate immune response or inflammation.

Immunosuppression: An act or event that reduces the activation orefficacy of the immune system. For example, immunosuppression isgenerally done to prevent the body from rejecting an organ transplant,treating graft-versus-host disease after a bone marrow transplant, orfor the treatment of autoimmune diseases such as rheumatoid arthritis orCrohn's disease. In an example, an agent including colutellin A isadministered to a subject in need of immunosuppression.

Inhibit: To decrease, limit or block the action or function of amolecule. In an example, the production of IL-2 is decreased, limited orblock by colutellin A. For example, the colutellin A inhibits, reducesor decreases IL-2 production, such as by a decrease of at least 10%, atleast 20%, at least 50%, at least 70%, or even at least 90%.

Mutant strain: A strain which arises spontaneously or through the effectof an external agent whether that agent is applied deliberately orotherwise.

Parental strain: The original endophytic Colletotrichum sp. strain, forinstance before mutagenesis (which leads to the mutated strain) orgenetic engineering (which leads to an engineered strain).

Rhizoctonia solani: A basidiomycete fungus which primarily attacks belowground plant parts such as the seeds, hypocotyls, and roots, but is alsocapable of infecting above ground plant parts (e.g., pods, fruits,leaves and stems). The most common symptom of Rhizoctonia disease isreferred to as “damping-off” characterized by non germination ofseverely infected seed whereas infected seedlings can be killed eitherbefore or after they emerge from the soil. Infected seedlings not killedby the fungus often have cankers, which are reddish-brown lesions onstems and roots.

Sclerotinia sclerotiorum: A fungus that is capable of infecting numerouscrops including lettuce, broccoli, cabbage, cauliflower, carrots,celery, beans, tomato, peppers, potatoes, stocks, sunflower, eggplant,squash, artichoke, asparagus, beet, broad bean, flower crops andlandscape shrubs. A typical initial symptom of S. sclerotiorum is thepresence of a cottony, white, dense mat of mycelial growth (mass offungus strands) on the surface of the host and on the adjacent soilsurface. Within this fluffy white mass, dense white bodies of fungussoon form. These bodies become black and hard as they mature and arecalled sclerotia. The sclerotia act like seeds and allow the fungus tosurvive for several years in the soil.

Subject: Living multi-cellular vertebrate organisms, a category thatincludes human and non-human mammals (such as laboratory or veterinarysubjects). In an example, a subject is a human. In an additionalexample, a subject is selected that is in need of immunosuppression.

Suppress (or decrease): To reduce the quality, amount, or strength ofsomething. In one example, a therapy suppresses or reduces an immuneresponse or one or more symptoms associated with an immune response, forexample as compared to the response in the absence of the therapy. In aparticular example, a therapy suppresses an immune response by at least10%, at least 20%, at least 50%, at least 70%, or even at least 90%.Such suppression can be measured using methods disclosed herein.

Substantially pure or Purified: A peptide, protein, or other activecompound that has been isolated from a cell, cell culture medium, orother crude preparation and subjected to fractionation to remove variouscomponents of the initial preparation, such as proteins, cellulardebris, and other components. Such purified preparations can includematerials in covalent association with the active agent, such asglycoside residues or materials admixed or conjugated with the activebiologically active agent, which may be desired to yield a modifiedderivative or analog of the active agent or produce a combinatorialtherapeutic formulation, conjugate, or the like. The term purified thusincludes such desired products as biologically active compounds whereinadditional compounds or moieties are bound to the biologically activeagent in order to allow for the attachment of other compounds and/orprovide for formulations useful in therapeutic treatment or diagnosticprocedures.

Generally, substantially purified peptides, proteins, or other activecompounds include more than 50%, for instance more than 80%, of allmacromolecular species present in a preparation prior to admixture orformulation of the respective compound with additional ingredients in acomplete pharmaceutical formulation for therapeutic administration.Additional ingredients can include a pharmaceutical carrier, excipient,buffer, absorption enhancing agent, stabilizer, preservative, adjuvantor other like co-ingredients. More typically, the peptide, protein, orother active compound is purified to represent greater than 90%, oftengreater than 95% of all macromolecular species present in a purifiedpreparation prior to admixture with other formulation ingredients. Inother cases, the purified preparation may be essentially homogeneous,wherein other macromolecular species (contaminants) are less than 1%. Inan example, a substantially pure Colletotrichum sp. compound is definedherein as a Colletotrichum sp. preparation (e.g., colutellin A(molecular mass of 1081.7), colutellin B (molecular mass of 1095.7),colutellin C (molecular mass of 1111.7) or colutellin D (molecular massof 1127.7)) which contains at least 80% of at least one of theseidentified compounds or a combination thereof, such as at least 85%, 90%or 95% of at least one of the Colletotrichum sp. compounds, including,but not limited to compounds with a molecular mass of 1081.7, 1095.7,1111.7, 1127.7 or a combination thereof.

T-Cell: A white blood cell critical to the immune response. T-cellsinclude, but are not limited to, CD4⁺ T cells and CD8⁺ T cells. A CD4⁺ Tlymphocyte is an immune cell that carries a marker on its surface knownas “cluster of differentiation 4” (CD4). These cells, also known ashelper T cells, help orchestrate the immune response, including antibodyresponses as well as killer T cell responses. CD8⁺ T-cells carry the“cluster of differentiation 8” (CD8) marker. In one embodiment, a CD8T-cell is a cytotoxic T lymphocyte. In another embodiment, a CD8 cell isa suppressor T-cell.

Therapeutically effective amount: An amount of an agent (such as anagent that includes colutellin A), that alone, or together with one ormore additional therapeutic agents, induces the desired response, suchas suppression of an immune response. In one example, it is an amount ofan agent including colutellin A needed to prevent or delay an immuneresponse, cause regression of an existing immune response or treat oneor more signs or symptoms associated with an immune response, in asubject. Ideally, a therapeutically effective amount provides atherapeutic effect without causing a substantial cytotoxic effect in thesubject. The preparations disclosed herein are administered intherapeutically effective amounts.

In an example, a desired response is to reduce or decrease an immuneresponse associated with an organ and tissue transplantation, variousautoimmune diseases, or certain non-autoimmune inflammatory diseases.For example, the agent can suppress the immune response by a desiredamount, for example by at least 5%, at least 10%, at least 15%, at least20%, at least 25%, at least 30%, at least 50%, at least 75%, or even atleast 90%, as compared to a response in the absence of the agent.

The effective amount of an agent that includes colutellin A, that isadministered to a human or veterinary subject will vary depending upon anumber of factors associated with that subject, for example the overallhealth of the subject. An effective amount of an agent can be determinedby varying the dosage of the product and measuring the resultingtherapeutic response, such as the suppression of the immune response.Effective amounts also can be determined through various in vitro, invivo or in situ immunoassays. The disclosed agents can be administeredin a single dose, or in several doses, as needed to obtain the desiredresponse. However, the effective amount of can be dependent on thesource applied, the subject being treated, the severity and type of thecondition being treated, and the manner of administration.

In particular examples, a therapeutically effective dose of an agentincluding colutellin A, colutellin B, colutellin C, colutellin D or acombination thereof includes at least 1 μg daily (such as 1-100 μg or5-50 μg) if administered via injection, or at least 1 mg daily ifadministered topically (such as 1-100 mg or 5-50 mg) of the agent thatincludes the desired colutellin. In particular examples, such dailydosages are administered in one or more divided doses (such as 2, 3, or4 doses) or in a single formulation.

The disclosed agents that include colutellin A can be administeredalone, in the presence of a pharmaceutically acceptable carrier, in thepresence of other therapeutic agents or both.

Under conditions sufficient for: A phrase that is used to describe anyenvironment that permits the desired activity. In one example, includestreating cells (such as cells infected with a target pathogen) with acompound including at least one of the disclosed Colletotrichum sp.isolates sufficient to allow the desired activity. In particularexamples, the desired activity is the inhibition of growth by therespective fungus. For example, the targeted pathogen is a fungus andthe desired activity is to inhibit or at least reduce the growth of thefungus. In another example, the target is a cell involved in mounting ormaintaining an immune response and the desired activity is to suppressor inhibit the immune response.

Unit Dosage Form: Physically discrete units suitable as unitary dosagesfor application to a subject (e.g., a human subject or animal, or aplant), each unit containing a predetermined quantity of active materialcalculated to produce the desired pharmaceutical effect in associationwith the required pharmaceutical diluent, carrier, or vehicle.

II. Overview of Several Embodiments

The current disclosure describes the isolation and identification of aC. dematium strain from Pteromischum sp. growing in a tropical forest inCosta Rica. This fungus is an endophyte that was identified on the basisof its morphological and genetic characteristics. Extracts of thefermentation broth of C. dematium possess selective antifungal activityassociated with a novel antimycotic peptide that the inventors termedcolutellin A. Characterization studies revealed that colutellin Ainhibited production of interleukin 2 (IL-2) from activated CD4⁺T-cells, suggesting that it may have potential as a novelimmunosuppressive drug. Surprisingly, in contrast to cyclosporin A, itpossessed little or no cytotoxicity to human blood cells. These findingswere reported in Microbiology 154: 1973-1979, 2008 which is herebyincorporated by reference in its entirety.

Based on these discoveries, disclosed herein is an endophytic fungusisolated from a Pteromischum sp. plant, wherein the isolated endophyticfungus has antifungal or immunosuppressive activity. In an example, theisolated endophytic fungus is a Colletotrichum sp. In one example, thefungus has biological activity against a fungal plant pathogen, such asBotrytis cinerea, Sclerotinia sclerotiorum, or Rhizoctonia solani. Forinstance, specifically the CR-12 strain of C. dematium is providedherein.

Also disclosed herein are crude extracts obtained from the endophyticfungus isolated from a Pteromischum sp. plant. In an example, the crudeextract is isolated from an endophytic fungus (e.g., C. dematium, suchas the CR-12 strain) that has biological activity against a fungal plantpathogen, such as Botrytis cinerea, Sclerotinia sclerotiorum, orRhizoctonia solani.

Compounds produced by the disclosed isolated endophytic fungus are alsoprovided. In an example, a compound has a molecular mass of 1081.7,1095.7, 111.7 or 1127.7. In some examples, a compound includes theN-terminal tetrapeptide sequence Val-Ile-Ser-Ile (SEQ ID NO: 1).

Also disclosed are methods of suppressing an immune response. In anexample, a method includes administering to a subject in need ofimmunosuppression a therapeutically effective amount of an agentincluding a disclosed endophytic fungus, crude extract isolated from adisclosed endophytic fungus or compound produced therefrom, therebysuppressing the immune response. In some examples, the method furtherincludes first selecting a subject in need of immunosuppression.

Methods to protect a plant against a fungal pathogen, such as Botrytiscinerea, Rhizoctonia solani, and/or Sclerotinia sclerotiorum areprovided. In an example, a method to protect a plant against a fungalpathogen includes treating the plant with an effective amount of adisclosed endophytic fungus, a crude extract isolated from a disclosedendophytic fungus or compound produced therefrom, thereby protecting theplant against the fungal pathogen. In some examples, the plant istreated by applying the strain directly to the plant. In other examples,the plant is treated by applying the strain to soil adjacent to theplant. In certain examples, the plant is treated by applying the strainto seed.

III. Isolated Endophytic Microorganisms

In the present disclosure, the isolation of specific endophyticColletotrichum sp. that produce antimycotic, immunosuppressive or otherbiologically active compounds of interest involves selecting one or moreplants as a source of the endophyte. In an example, the selectionprocess is conducted on the basis of the environment, age, or naturalhistory of a given plant. Such selection methods involve culturingtissue from the interior region of a dicotyledonous plant, e.g.,Pteromischum sp. plant, on nutrient media for a time sufficient topermit colony formation by a strain of endophytic Colletotrichum sp.associated with the plant tissue and selecting one or more endophyticColletotrichum sp. strains demonstrating the biological activity ofinterest. Various means can be used to select the endophyticColletotrichum sp. isolates, and the isolates can be tested through anyof numerous methods known in the art to discover a biological activityof interest, either by measuring some activity of the isolates directly,e.g., by zones of inhibition, or by preparing and testing extracts orpurified compounds from the isolates.

In an example, the biological activity of interest can inhibit orsuppress an immune response. In another example, the biological activityof interest can be to control or inhibit plant pathogens. For example,the biological activity is against fungi (such as Botrytis cinerea,Sclerotinia sclerotiorum, and Rhizoctonia solani).

In an example, endophytic Colletotrichum sp. isolates are isolated froma Pteromischum sp. For example, endophytic Colletotrichum sp. isolatescan be isolated by fermentation and the broth extracted with an organicsolvent, e.g., n-butanol, and the contents of the residue purified bybioassay guided high performance liquid chromatography using the fungusBotrytis cinerea as the test organism. In an example, a compound with amolecular mass of 1081.7, 1095.7, 1111.7, or 1127.7 is obtained.

IV. Biologically Active Agents

The present disclosure relates in certain embodiments to biologicallyactive agents useful in treating or preventing various conditions. Thebiologically active agents in various examples are the Colletotrichumsp. isolates themselves, crude extracts obtained by cultivating suchisolates under culture conditions, or compounds (such as peptides)isolated from the isolates. In an example, the biologically active agentincludes at least one compound with a molecular mass of 1081.7, 1095.7,1111.7, and 1127.7 or a combination of such compounds. For example, abiologically active agent includes colutellin A peptide (a cycliclipopeptide with a molecular mass of 1127.7) and possesses bioactivitiesat least against fungi (such as Botrytis cinerea and Sclerotiniasclerotiorum) as well as immunosuppressive activities (includinginhibiting the production of IL-2 from CD4). In this manner, thedisclosure also provides novel biologically active extracts, compounds(e.g., a family of cyclic lipopeptides) and agents.

V. Uses of Biologically Active Compounds and Agents

The biologically active compounds of the present disclosure can be usedto suppress an immune response or to control a pathogenic organism, suchas a fungus. The agent, including one of the disclosed compounds, isprovided in an amount effective to inhibit the pathogenic organism orcondition for a time and under conditions permitting the agent toinhibit the pathogenic organism or condition.

The Colletotrichum sp. isolates have significant immunosuppressive andantifungal activity. In an example, the biologically active agents canbe used to suppress an immune response. For example, a biologicallyactive agent including an endophytic Colletotrichum sp. compound with amolecular mass of 1081.7, 1095.7, 1111.7, and 1127.7 or a combination ofsuch compounds is employed to suppress an immune response associatedwith an organ or tissue transplantation, autoimmune disease, or anon-autoimmune inflammatory disease. In a particular example, abiologically active agent including colutellin A (molecular mass of1127.7) is utilized to suppress an immune response, such as oneaffiliated with an organ or tissue transplantation, autoimmune disease,or a non-autoimmune inflammatory disease.

In a further example, the biologically active agents can be used tocontrol diverse fungal pathogens including, but not limited to,Rhizoctonia solani, Botrytis cinerea, and Sclerotinia sclerotiorum. Forexample, a biologically active agent including an endophyticColletotrichum sp. compound such as colutellin A, colutellin B,colutellin C, colutellin D or a combination of such compounds isemployed to control fungi including Rhizoctonia solani, Botrytiscinerea, and Sclerotinia sclerotiorum. In a particular example, abiologically active agent including colutellin A (molecular mass of1127.7) is utilized to control fungi, such as Rhizoctonia solani,Botrytis cinerea, and Sclerotinia sclerotiorum. For example, thebiologically active agents including an endophytic Colletotrichum sp.compound (such as an endophytic Colletotrichum sp. compound with amolecular mass of 1081.7, 1095.7, 1111.7, and 1127.7 or a combination ofsuch compounds) can be used to treat or protect plants challenged orinfected by any myriad of plant pathogens, such as pathogenic fungi, andmay be used to treat diseases in the field, soil or in post harvestapplications. Additional agricultural applications include, but are notlimited to, treatment in seed coats, on agricultural implements, leaf orplant surfaces, and building or other material surfaces—generally, anysite which may contain or come into contact with a pathogen.

VI. Methods of Production

The present disclosure also relates to methods for producing thedescribed biological agents. The biological agents may be an endophyticColletotrichum sp.; an extract of the endophytic Colletotrichum sp.; ora compound (e.g., a cyclic lipopeptide) obtained from the endophyticColletotrichum sp., e.g., compound with a molecular mass of 1081.7,1095.7, 1111.7, and 1127.7 or a combination of the compounds thereof,having the biological activity of interest. Representative methodsinclude cultivating an isolate of an endophytic Colletotrichum sp. andrecovering the cells or a biological agent from the culture medium. Itmay be desirable thereafter to form the free acid or a salt or ester bymethods known by one of ordinary skill in the art.

The endophytic Colletotrichum sp., or a high yielding or otherwisemodified mutant thereof, may be used in the methods of the presentdisclosure to produce biologically active agents.

In an example, the endophytic Colletotrichum sp. is cultivated in anutrient medium suitable for production of the heterologous biologicalsubstance using methods known in the art. For example, the cell may becultivated by shake flask cultivation, small-scale or large-scalefermentation (including continuous, batch, fed-batch, or solid statefermentations) in laboratory or industrial fermentors performed in asuitable medium and under conditions allowing the biological substanceto be expressed and/or isolated. The cultivation takes place in asuitable nutrient medium comprising carbon and nitrogen sources andinorganic salts, using procedures known in the art. Suitable media areavailable from commercial suppliers or can be prepared according topublished compositions (e.g., in catalogues of the American Type CultureCollection).

In one example, the nutrient media for the cultivation of the endophyticColletotrichum sp. contains, in the range of about 0.1 to about 10%, acomplex organic nitrogen source such as yeast extract, corn steepliquor, vegetable protein, seed protein, hydrolysates of such proteins,milk protein hydrolysates, fish and meat extracts, and hydrolysates suchas peptones. In an alternative example, chemically defined sources ofnitrogen can be used such as urea, amides, single or mixtures of commonamino acids such as valine, asparagine, glutamic acid, proline, andphenylalanine. In further examples, carbohydrates (0.1-5%) are includedin the nutrient media and starch or starch hydrolysates such as dextrin,sucrose, lactose or other sugars or glycerol or glycerol esters may alsobe used. The source of carbon can be derived from vegetable oils oranimal fats. Carboxylic acids and their salts can be included as asource of carbon for growth and production of β-lactamase inhibitors. Aparticularly suitable low cost medium is one containing soy bean flourplus dried malt distillers solubles plus dextrin.

In an example, mineral salts such NaCl, KCl, MgCl₂, ZnCl₂, FeCl₃, Na_(e)SO₄, FeSO₄, MgSO₄ and Na⁺ or K⁺ salts of phosphoric acid are added tothe media described above particularly if chemically defined. In furtherexamples, CaCO₃ (as a source of Ca⁺⁺ ions or for its buffering action),salts of trace elements (such as nickel, cobalt or manganese) orvitamins are added to the media.

The present disclosure is also directed to a mutant of an endophyticColletotrichum sp. wherein the amount of the biological activity agentproduced by the mutant is greater than the amount of the substanceproduced by a corresponding parental strain. The present disclosure isfurther directed to methods for obtaining such a mutant. In one example,an endophytic Colletotrichum sp. is obtained from a mutant of anendophytic Colletotrichum sp. strain, wherein the substance is producedin an amount greater than the amount of the substance produced by acorresponding parental strain. Suitable methods of producing mutantstrains are well-known to those in the art, and include, for example,ionizing radiation (such as gamma-rays or X-rays), UV light, UV lightplus a photosensitizing agent (such as 8-methoxypsoralen), nitrous acid,hydroxylamine, purine or pyrimidine base analogues (such as5-bromouracil and N-methyl-N′-nitro-N-nitrosoguanidine), acridines,alkylating agents (such as mustard gas, ethyl-methane sulphonate),hydrogen peroxide, phenols, formaldehyde, and heat. Alternatively,mutants may be produced through genetic techniques such asrecombination, shuffling, transformation, transduction, lysogenisation,lysogenic conversion, and selective techniques for spontaneous mutants.Specifically, one method of mutating an endophytic Colletotrichum sp.strain and selecting such a mutant comprises the following procedure:(i) the parental strain is treated with a mutagen; (ii) the thuspresumptive mutants are grown in a medium suitable for selection of amutant strain; and (iii) the mutant strain is selected on the basis ofincreased production of a compound of the present disclosure. In aspecific example, the selected colonies are grown in a normal productionmedium, and a final selection for such mutants is performed.

The present disclosure also relates to methods for obtaining a“substantially pure” endophytic Colletotrichum sp. compound, such as anendophytic Colletotrichum sp. compound which contains less than 5%contaminants. For example, the substantially pure endophyticColletotrichum sp. compound contains at least 95% of one of thedisclosed compounds (a compound with a molecular mass of 1081.7, 1095.7,1111.7, and 1127.7 or combination thereof). In an example, endophyticColletotrichum sp. isolate, or other compounds of endophyticColletotrichum sp., are extracted from the culture filtrate by a varietyof methods known to the art. In a specific example, the cells of theendophytic Colletotrichum sp. are first removed from the fermentation byfiltration or centrifugation before such extraction procedures arecommenced. Precipitation by solvent extraction from culture filtrate,which may use an adjusted to acid pH values and methods based on theanionic nature of the metabolite such as the use of anion exchangeresins can be utilized. Other primary methods of isolation which may beused include conventional methods such as adsorption onto carbon,precipitation, salting out, molecular filtration, or any method known inthe art.

VII. Compositions

The present disclosure also relates to compositions comprising abiological agent as described herein. The biological agent may be anendophytic Colletotrichum sp., an extract of the endophyticColletotrichum sp., or a compound, such as a cyclic lipopeptide,obtained from the endophytic Colletotrichum sp., e.g., a compound with amolecular mass of 1081.7, 1095.7, 1111.7, and 1127.7 (such as colutellinA, B, C, and D) or a combination thereof, having the biological activityof interest. The composition can include a suitable carrier, or maycomprise the agent affixed to a substrate. The compositions including abiologically active agent of the present disclosure can be used tocontrol a range of pathogenic organisms (such as fungi), diseases, orconditions. The composition can also find use as applied to a substrate.The agent is provided in an amount effective to inhibit the pathogenicorganism or condition for a time and under conditions permitting theagent to inhibit the pathogenic organism or condition. Differentcompositions will be required for administration to plants, humans andanimals in unit dosage forms, such containing suitable quantities of thecompounds.

Common carriers and excipients include, but are not limited to, cornstarch or gelatin, lactose, sucrose, microcrystalline cellulose, kaolin,mannitol, dicalcium phosphate, sodium chloride, and alginic acid.

The endophytic Colletotrichum sp. isolate or other compounds, or a saltor ester thereof, obtainable from an endophytic Colletotrichum sp. canbe formulated into a pharmaceutical composition, which comprises thecompound, together with a pharmaceutically acceptable carrier.

The compound may be in the form produced by the endophyticColletotrichum sp., or the result of further chemical modification, forinstance to reduce toxicity and perhaps to increase efficacy oravailability (e.g., availability in a biological system, such as asubject). This approach has been effectively taken with antibioticfamily, obtained from a plant associated microbe—Pseudomonas syringae,namely, the pseudomycins (Ballio et al., FEBS Letters 355, 96-100,1994). A specific pseudomycin has been subjected to modifications byorganic synthesis and has yielded a derivative that is no longer toxicto mammalian systems and yet remains effective against human pathogenicfungi (Zhang et al., Bioorg. Med. Chem. Lett. 11, 123-126, 2001; Zhanget al., Bioorg. Med. Chem. Lett. 11, 903-907, 2001).

VIII. Administration of Compositions

The pharmaceutical compositions of the disclosure include those in aform adapted for oral, topical, or other potential use, and may be usedfor immunosuppression in animals, particularly mammals including but notlimited to humans.

Examples of suitable unit dosage forms in accord with the presentdisclosure are tablets, capsules, pills, suppositories, powder packets,wafers, granules, cachets, teaspoonfuls, tablespoonfuls, dropperfuls,ampoules, suspensions, syrups, vials, aerosols with metered discharges,segregated multiples of any of the foregoing, and other forms as hereindescribed. Such compositions may contain conventional pharmaceuticallyacceptable materials such as diluents, binders, colors, flavors,preservatives, disintegrants and the like in accordance withconventional pharmaceutical practice in the manner well understood bythose skilled in the art of formulating pharmaceutical compounds. Theconcentration of a compound in the unit dosage may vary, for example,from about 1 percent to about 50 percent depending on the particularform of the compound and its solubility and the dose desired.

For oral administration, either solid or fluid unit dosage forms can beprepared. For preparing solid compositions such as tablets, the desiredcompound is mixed with conventional ingredients such as talc, magnesiumstearate, dicalcium phosphate, magnesium aluminum silicate, calciumsulfate, starch, lactose, acacia, methylcellulose, and functionallysimilar materials as pharmaceutical diluents or carriers. Disintegratorscommonly used in the compositions of the disclosure includecroscarmellose, microcrystalline cellulose, corn starch, sodium starchglycolate, and alginic acid. Capsules are prepared by mixing thecompound with an inert pharmaceutical diluent and filling the mixtureinto a hard gelatin capsule of appropriate size. Soft gelatin capsulesare prepared by machine encapsulation of a slurry of the compound withan acceptable vegetable oil, light liquid petrolatum, or other inertoil.

Fluid unit dosage forms for oral administration such as syrups, elixirs,and suspensions can also be prepared. The water-soluble forms can bedissolved in an aqueous vehicle together with sugar, aromatic flavoringagents and preservatives to form a syrup. An elixir is prepared by usinga hydroalcoholic (ethanol) vehicle with suitable sweeteners such assugar and saccharin, together with an aromatic flavoring agent.

Suspensions can be prepared with an aqueous vehicle with the aid of asuspending agent such as acacia, tragacanth, methylcellulose, and thelike.

Tablet binders that can be included are acacia, methylcellulose, sodiumcarboxymethylcellulose, poly-vinylpyrrolidone (Povidone), hydroxypropylmethylcellulose, sucrose, starch and ethylcellulose.

Lubricants that can be used include magnesium stearate or other metallicstearates, stearic acid, silicone fluid, talc, waxes, oils, andcolloidal silica.

Flavoring agents such as peppermint, oil of wintergreen, cherryflavoring, or the like can also be used. It may be desirable to add acoloring agent to make the dosage form more attractive in appearance orto help identify the product.

For parenteral administration, fluid unit dosage forms are preparedutilizing the compound and a sterile vehicle, with water beingpreferred. The compound, depending on the vehicle and concentrationused, can be either suspended or dissolved in the vehicle. In preparingsolutions, the compound can be dissolved in water for injection andfiltered sterilized before filling into a suitable vial or ampoule andsealing. Advantageously, adjuvants such as a local anesthetic,preservative, and buffering agents can be dissolved in the vehicle. Toenhance the stability, the composition can be frozen after filling intothe vial and the water removed under vacuum. The dry lyophilized powderis then sealed in the vial and an accompanying vial of water forinjection is supplied to reconstitute the liquid prior to use.Parenteral suspensions can be prepared in substantially the same mannerexcept that the compound is suspended in the vehicle instead of beingdissolved and sterilization cannot be accomplished by filtration. Thecompound can be sterilized by exposure to ethylene oxide beforesuspending in the sterile vehicle. Advantageously, a surfactant orwetting agent is included in the composition to facilitate uniformdistribution of the compound.

Additionally, a rectal suppository can be employed to deliver thecompound. This dosage form is of particular interest where the mammalcannot be treated conveniently by means of other dosage forms, such asorally or by insufflation, as may be the case of animals, or youngchildren, or debilitated persons. The compound can be incorporated intoany of the known suppository bases using methods known in the art.Examples of such bases include cocoa butter, polyethylene glycols(carbowaxes), polyethylene sorbitan monostearate, and mixtures of thesewith other compatible materials to modify the melting point ordissolution rate. These rectal suppositories can weigh from about 1 to2.5 gm.

Typically, any effective quantity of a compound of the presentdisclosure is employed in treatment. The determination of an appropriatedosage of the compound for a given treatment depends on many factorsthat are well known to those skilled in the art. They include forexample, the route of administration and the potency of the particularcompound.

The particular compound may be present in the composition as the soletherapeutic agent or may be present together with other therapeuticagents, either related or unrelated to the original compound.

A convenient method of practicing the treatment method may be toadminister a compound of the present disclosure via intravenous (IV)infusion. In this procedure, a sterile formulation of a suitable solublesalt of the compound is incorporated in a physiological fluid, such as5% dextrose solution, and the resulting solution is infused slowly by IVadministration. Alternatively, the piggy-back method of IV infusion canalso be used. For IV use, a water soluble form of the antibiotic can bedissolved in one of the commonly used intravenous fluids andadministered by infusion. Such fluids as, for example, physiologicalsaline, Ringer's solution, or 5% dextrose solution can be used.

For intramuscular preparations, a sterile formulation of a suitablesoluble salt form of the compound, for example the hydrochloride salt,can be dissolved and administered in a pharmaceutical diluent such aspyrogen-free water (distilled), physiological saline or 5% glucosesolution. A suitable insoluble form of the compound may be prepared andadministered as a suspension in an aqueous base or a pharmaceuticallyacceptable oil base, for example, an ester of a long chain fatty acidsuch as ethyl oleate.

A composition comprising a compound of the present disclosure (e.g., anendophytic Colletotrichum sp. isolate, such as a cyclic lipopeptide) canbe administered in a single daily dose or in multiple doses per day. Thetreatment regimen may require administration over extended periods oftime, for example, for several days or weeks (such as for one to sixweeks). The amount per administered dose or the total amountadministered will depend on such factors as the nature and severity ofthe infection, the age and general health of the patient, the toleranceof the patient to the compound.

IX. Exemplary Uses of Compositions

Compositions as described herein may be used for immunosuppression or totreat a fungal infection in an organism, such as a plant.

Suppression of an Immune Response

Provided, then, are compositions and methods for suppressing an immuneresponse in an organism (e.g., mammal), which comprises administering tothe organism a therapeutically effective amount of an endophyticColletotrichum sp. isolate, such isolate C-12, a compound (such as acyclic lipopeptide (e.g., colutellin A)), or a salt or ester thereof.For example, the composition including endophytic colutellin A is usefulfor suppressing an immune response associated with an organ or tissuetransplantation, autoimmune disease, or a non-autoimmune inflammatorydisease. For example, the composition including endophytic colutellin A,B, C, D or a combination thereof suppresses the immune response by atleast 10%, such as at least 20%, at least 30%, at least 50%, or at least70%.

Exemplary autoimmune diseases affecting mammals include rheumatoidarthritis, juvenile oligoarthritis, collagen-induced arthritis,adjuvant-induced arthritis, Sjogren's syndrome, multiple sclerosis,experimental autoimmune encephalomyelitis, inflammatory bowel disease(e.g., Crohn's disease, ulcerative colitis), autoimmune gastric atrophy,pemphigus vulgaris, psoriasis, vitiligo, type 1 diabetes, non-obesediabetes, myasthenia gravis, Grave's disease, Hashimoto's thyroiditis,sclerosing cholangitis, sclerosing sialadenitis, systemic lupuserythematosis, autoimmune thrombocytopenia purpura, Goodpasture'ssyndrome, Addison's disease, systemic sclerosis, polymyositis,dermatomyositis, autoimmune hemolytic anemia, pernicious anemia, and thelike. Further, exemplary inflammatory diseases affecting mammals includerheumatoid arthritis, osteoarthritis, inflammatory lung disease(including chronic obstructive pulmonary lung disease), inflammatorybowl disease (including ulcerative colitis and Crohn's Disease),periodontal disease, polymyalgia rheumatica, atherosclerosis, systemiclupus erythematosus, systemic sclerosis, Sjogren's Syndrome, asthma,allergic rhinitis, and skin disorders (including dermatomyositis andpsoriasis) and the like. In particular examples, a therapeuticallyeffective amount of an endophytic Colletotrichum sp. isolate, suchisolate C-12, a compound (such as a cyclic lipopeptide (e.g., colutellinA)), or a salt or ester thereof is administered to suppress suppressingan immune response associated with at least or combination thereof ofthe aforementioned diseases. The method includes administering to thesubject an amount of a compound of the present disclosure which iseffective for this purpose. In general, an effective amount is a dosebetween about 0.5 and about 100 mg/kg. A particular dose is from about 1to about 60 mg/kg of active compound. A typical daily dose for an adulthuman is from about 1 mg to about 50 mg.

Treatment of Fungal Infections

Also provided are compositions and methods of treating fungal infectionin an organism, such as a plant or mammal, which comprises administeringto the organism an anti-fungal effective amount of an endophyticColletotrichum sp. compound, such as a cyclic lipopeptide (e.g.,colutellin A), or a salt or ester thereof. The compositions can also beused to control diverse fungal pathogens including, but not limited to,Rhizoctonia solani, Botrytis cinerea, and Sclerotinia sclerotiorum.

The compositions of the disclosure may be pesticidal compositions usedfor administration to plants, or the associated soil, equipment,containers, machinery, surfaces and so forth. For use with a plant, themethod of use may involve applying an endophytic Colletotrichum sp.strain, or an extract or compound (e.g., peptide) derived from thestrain either directly to the plant, or to soil adjacent to the plant.In some cases the treatment may be made to seeds, e.g., in the format ofseed coats, soaks, or other such applications. In certain circumstances,the strain (rather than an extract) can be applied to grow inassociation with the plant and produce biologically active compoundscapable of protecting the plant against plant pathogen attack, such asfungal attack.

The present disclosure is further directed to pesticidal compositionscomprising the substance in an effective amount to control a pest, and apesticidal carrier. For example, an effective amount is the amount ofthe substance sufficient to control a pest through killing or stuntingof the growth of the pest or protecting a plant from pest infestation.The pesticidal compositions may comprise a compound of the presentdisclosure in a substantially pure form or as an extract from a wholebroth culture of an endophytic Colletotrichum sp. in dry, concentrated,or liquid form and a suitable pesticidal carrier, examples of which aredisclosed infra. The substance is present in the composition at aconcentration of about 0.001% to about 60% (w/w).

The pesticidal compositions may further comprise a deposition agentwhich assists in preventing the composition from drifting from thetarget area during application (e.g., as it is sprayed from a plane), orfrom being blown away from the plant once it has been deposited. Thedeposition agent in the compositions of the present disclosure ispreferably a proteinaceous material, which has the added benefit ofbeing palatable to the insect. Any animal or vegetable protein issuitable for this purpose, in dry or in liquid form. Examples of usefulsources of protein which can be conveniently and economically added tothe composition include, but are not limited to, soy protein, potatoprotein, soy flour, potato flour, fish meal, bone meal, yeast extract,and blood meal. Alternative deposition agents include modified cellulose(carboxymethylcellulose), botanicals (grain flours, ground plant parts),non-phyllosilites (talc, vermiculite, diatomaceous earth), natural clays(attapulgite, bentonite, kaolinite, montmorillonite), and syntheticclays (Laponite). When utilized, the deposition agent is present in thepesticidal compositions of the present disclosure in an amount ofbetween about 0.4% w/w and about 50% w/w, preferably between about 1%w/w and about 20% w/w.

The pesticidal compositions may further comprise an antifreeze/humectantagent which suppresses the freeze point of the product and helpsminimize evaporation when sprayed and which maintains deposit texturemaking the product more efficacious and palatable. Examples ofantifreeze/humectant agents include, but are not limited to, ethyleneglycol, propylene glycol, dipropylene glycol, glycerol, butyleneglycols, pentylene glycols and hexylene glycols. When utilized, theantifreeze/humectant agent is present in the pesticidal compositions ofthe present disclosure in an amount of between about 0.5% w/w and about25% w/w, preferably between about 2% w/w and about 15% w/w.

The pesticidal compositions may further comprise a surfactant in anamount where it acts as an emulsifying, a wetting, or a dispersingagent. Examples of such surfactants are anionic surfactants such ascarboxylates, for example, a metal carboxylate of a long chain fattyacid; N-acylsarcosinates; mono or di-esters of phosphoric acid withfatty alcohol ethoxylates or salts of such esters; fatty alcoholsulphates such as sodium dodecyl sulphate, sodium octadecyl sulphate orsodium cetyl sulphate; ethoxylated fatty alcohol sulphates; ethoxylatedalkylphenol sulphates; lignin sulphonates; petroleum sulphonates; alkylaryl sulphonates such as alkyl-benzene sulphonates or loweralkylnaphthalene sulphonates, e.g., butyl naphthalene sulphonate; saltsor sulphonated naphthalene-formaldehyde condensates; salts ofsulphonated phenol-formaldehyde condensates; or more complex sulphonatessuch as amide sulphonates, e.g., the sulphonated condensation product ofoleic acid and N-methyl taurine or the dialkyl sulphosuccinates, e.g.,the sodium sulphonate or dioctyl succinate. Further examples of suchsurfactants are non-ionic surfactants such as condensation products offatty acid esters, fatty alcohols, fatty acid amides or fatty-alkyl- oralkenyl-substituted phenols with ethylene oxide, block copolymers ofethylene oxide and propylene oxide, acetylenic glycols such as2,4,7,9-tetraethyl-5-decyn-4,7-diol, or ethoxylated acetylenic glycols.Further examples of such surfactants are cationic surfactants such asaliphatic mono-, di-, or polyamine as acetates, naphthenates or oleates;oxygen-containing amines such as an amine oxide of polyoxyethylenealkylamine; amide-linked amines prepared by the condensation of acarboxylic acid with a di- or polyamine; or quaternary ammonium salts.When utilized, the surfactant is present in an amount of between about0.5% w/w and about 25% w/w, preferably between about 1% w/w and about 8%w/w.

The pesticidal compositions may further comprise an inert material.Examples of inert materials include inorganic minerals such asdiatomaceous earth, kaolin, mica, gypsum, fertilizer, phyllosilicates,carbonates, sulfates, or phosphates; organic materials such as sugars,starches, or cyclodextrins; or botanical materials such as woodproducts, cork, powdered corncobs, rice hulls, peanut hulls, and walnutshells.

The pesticidal compositions may further comprise a preservative, afeeding stimulant, an attractant, an encapsulating pesticide, a binder,a dye, an ultraviolet light protectant, a buffer, a flow agent, or othercomponent to facilitate product handling and application for particulartarget pests.

The pesticidal compositions can be applied in a dry or liquid form,e.g., a suspension, a solution, an emulsion, a dusting powder, adispersible granule, a wettable powder, an emulsifiable concentrate, anaerosol or impregnated granule, or a concentrate or primary compositionwhich requires dilution with a suitable quantity of water or otherdiluent before application. The concentrations of each component in thecomposition will vary depending upon the nature of the particularcomposition, specifically, whether it is a concentrate or to be useddirectly. The composition may contain about 1% to about 98% of a solidor liquid inert carrier. The compositions will be preferablyadministered at the labeled rate for commercial products, preferablyabout 0.01 pound to 5.0 pounds per acre when in dry form and at about0.01 pint to 25 pints per acre when in liquid form.

The pesticidal compositions can be applied directly to a plant by, forexample, spraying or dusting at the time when the pest has begun toappear on the plant or before the appearance of pests as a protectivemeasure. The pesticidal compositions can be applied by foliar, furrow,broadcast granule, “lay-by”, or soil drench application. Thecompositions can also be applied directly to ponds, lakes, streams,rivers, still water, and other areas subject to infestation by pests ofconcern to public health. The compositions can be applied by spraying,dusting, sprinkling, or the like. The spray or dust can convenientlycontain another pesticide.

The pesticidal compositions can be applied to protect a number ofdifferent plant types, including, but not limited to, cereals (wheat,barley, rye, oats, rice, sorghum and related crops), beets (sugar beetand fodder beet), drupes, pomes and soft fruit (apples, pears, plums,peaches, almonds, cherries, strawberries, raspberries, andblackberries), leguminous plants (alfalfa, beans, lentils, peas,soybeans), oil plants (rape, mustard, poppy, olives, sunflowers,coconuts, castor oil plants, cocoa beans, groundnuts), cucumber plants(cucumber, marrows, melons), fiber plants (cotton, flax, hemp, jute),citrus fruit (oranges, lemons, grapefruit, mandarins), other fruits(such as bananas, pineapples, cassayas, mangos, guavas, grapes, and soforth), vegetables (spinach, lettuce, asparagus, cabbages and otherbrassicae, carrots, onions, tomatoes, potatoes), lauraceae (avocados,cinnamon, camphor), deciduous trees and conifers (linden-trees,yew-trees, oak-trees, alders, poplars, birch-trees, firs, larches,pines), or plants such as maize, turf plants, tobacco, nuts, coffee,sugar cane, tea, vines, hops, and natural rubber plants, as well asornamental plants as well as ornamental plants and particularly plantswhich are grown for their flowers. It will be appreciated that thelisted plants are representative only, rather than limiting.

The present disclosure is further described by the following examples,which should not be construed as limiting the scope of the disclosure.

Example 1 Materials and Methods

This example provides a description of the material and methods utilizedfor the isolation and characterization of Colletotrichuin sp.

Isolating, culturing, identifying, and storing of Colletotrichum sp. Theculture of Colletotrichum sp. was obtained as an endophyte from a smallcutting made on an immature Pteromischum sp. plant collected in aCaribbean costal Costa Rican rainforest. A number of other plantsamples, including Dipteryx sp., Monstera sp. and Cercopia sp and otherswere collected at the same time and in the same area. Each plant samplewas given a numerical designation. Endophytes were recovered from eachplant made in the collection using the standard methods of surfacetreatment, tissue removal and plating on water agar (Strobel & Daisy,Microbiol. Mol. Biol. Rev. 67: 491-502, 2003). One fungus, designatedCR-12, was recovered from Pteromischum sp. and when grown on potatodextrose agar (PDA) was initially shown to have antimycotic activity byvirtue of a bioassay test (Castillo et al., Microbial Ecol. 53: 12-19,2007). The organism was examined for its morphological and spore-formingfeatures as described below for taxonomic purposes. In addition,molecular biological studies were performed on this fungus. It was grownon PD broth for 7 days and the mycelium was harvested and the DNA wasextracted using the DNeasy Plant and Fungi Mini kit (QIAGEN®) accordingto the manufacturer's directions. The ITS regions of the fungus wereamplified using PCR and the universal ITS primers ITS 1 (59-TCC GTA GGTGAA CCT GCG G-39; SEQ ID NO: 2) and ITS4 (59-TCC TCC GCT TAT TGA TATGC-39; SEQ ID NO: 3). All other procedures were carried out as describedby Ezra et al. (Microbiology 150: 4023-4031, 2004). Sequence data weredeposited in GenBank (GenB ank Accession No. EU330193).

Plugs containing the mycelium were placed in 15% glycerol and stored at−70° C. However, the other storage conditions for the fungus wereobtained by growing the fungus on sterilized barley and placing theinfested grains at −70° C. The fungus was deposited as No. 2341 in theliving mycological culture collection at Montana State University.

Test Fungi and Bacteria.

All plant pathogenic fungi used in the bioassay test system wereobtained from Drs. Don Mathre and Nina Zidak of the MSU Department ofPlant Sciences. All fungi were grown on potato dextrose agar (PDA) at23° C. and only freshly transferred cultures (4-7 days old) were used inthe fungal bioassay tests.

Scanning Electron Microscopy (SEM).

Isolate CR-12 was grown on PDA and later processed for SEM. Many agarpieces containing the fungus were placed into filter paper packets andsuspended in 2% glutaraldehyde in 0.1 M sodium cacodylate buffer (pH7.2-7.4) with Triton X (a wetting agent). Tissues were aspirated for 5min. and incubated overnight as previously described (Ezra et al.,Microbiol. 150: 4023-4031, 2004). Ultimately, for SEM some of the fungalmaterial was critical point dried, gold sputter coated and images wererecorded with an FBI XL30 ESEM FEG in high vacuum mode using theEverhart-Thornley detector. Freshly prepared wet specimens were examinedby environmental scanning electron microscopy (ESEM) and images wererecorded with an FEI XL30 ESEM FEG in the environmental mode asdescribed by Castillo et al. (Scanning 27: 305-311, 2005). A gaseouselectron detector was used with a spot size of 3, at 15 kV. Thetemperature was 40° C. with a chamber pressure which ranged from 5 to 6Torr providing humidity up to 100% at the sample. Conidia were measuredusing Image J software (available online at web addressrsb.info.nih.gov/ij/).

Minimum Inhibitory Concentrations (MICs).

Assays were performed in sterile 24-well plates with each wellcontaining 500 μl of potato dextrose broth. The plates were incubatedfrom 48-288 hours at 25 C. The MIC was defined as the minimumconcentration of compound resulting in no visible growth of the testorganism. The compounds were dissolved in methanol which representedless than 0.5% total methanol in each test well. Several small plugs ofagar 3×3×3 mm containing actively growing test fungi were then placedinto each well with some wells serving as controls.

Bioassay Guided Isolation and Purification of Colutellin.

Colletotrichum sp. (171) was grown in shake culture for 28 days at 25°C. The fungal mycelium was removed from the fermentation broth byfiltration and extracted three times with equal volumes of n-butanol.The bioactivity of the extract/fraction was evaluated by placing a smallamount of material on a PDA plate and challenging with several plugs ofagar supporting the growth of Botrytis cinerea (bioassay guidedfractionation). Approximately 40 g (dry wt) of the extract wasfractionated by liquid chromatography on a 5.0×28.0 cm column of silicagel (Selecto Scientific—particle size 32-63) using 700 ml of eachsolvent system in a stepwise gradient of increasing polarity (A)methylene chloride 100%; (B) chloroform 100%; (C) chloroform:ethylacetate 50:50 v/v; (D) ethyl acetate 100%; (E) ethyl acetate:ethanol50:50 v/v; (F) ethanol 100% and (G) methanol 100%. Three fractions (B,C, D) were the most active in the antifungal assay (100 mg/ml). Theseactive fractions were flash evaporated to dryness and were againchromatographed on a 3.0×58.0 cm silica column (same material and samesolvent program) eluted with 1 l of each of the solvent systems A, B, C,D to obtain 8 active subfractions (ca. 400 ml each) using an identicalelution profile as before. The sub-fraction at 1.3-1.6 l was obtained byvirtue of its bioactivity against B. cinerea. It was further purified bysemipreparative HPLC on a Waters 600E HPLC with a Phenomenex Sphereclonecolumn 5 pODS (250×10 mm) under gradient conditions (flow: 5 ml min⁻¹, 0min H₂O:methanol 50:50 v/v; 20 min methanol 100%; 40 min acetonitrile100%). Detection was at 220 nm and the most biologically active producteluted in a distinct single peak at 24.6 min and it yielded only onecompound with a mass of 1127.7 (colutellin A). This compound was usedfor all biological assays and chemical analysis. However, otherbiological activity remained before and after this peak (broad peak) at1.1-1.91 as sub-fractions of the second silica column and it too wassubjected to HPLC and the main peak had a retention time of 23.9 min, itpossessed colutellin A and 3 other colutellin A-like derivatives. Thispeak was subjected to LC/MS analysis.

General Instrumental Procedures.

Ultraviolet (UV) spectra were recorded in 100% methanol using a BeckmanDU-50 UV-visible spectrophotometer. Spectra by NMR were recorded on aVarian INOVA AS-600 MHz spectrometer, using the signals of the residualsolvent protons as internal references (δ_(H) 3.3 and δ_(H) 4.9 ppm fordeuterated MeOH) at 23° C. Masses were determined on an LTQ FT ULTRA(Thermo Scientific). Samples were suspended in 50% (v/v) acetonitrile,0.1% (v/v) formic acid at a concentration of 10 pmol/μl, and introducedinto the instrument by nanoelectrospray at a flow rate of 50 nl/min.Tandem mass spectrometry and desalting studies were done on a QSTARsystem from Applied Biosystem (QqTOF). For desalting, measurements weretaken before and after sample application to a ZipTip® C18 (MilliporeCorp.). MALDI-TOF studies were performed on a Vayager DESTR MALDI-TOFinstrument (Applied Biosystems). Samples and matrix(α-cyano-4-hydroxycinnamic acid) were mixed at a ratio of 1:1 (v/v)before being spotted onto the MALDI plate and air dried. Electrospraymass spectral data also were acquired using a Micromass LCT TOF massspectrometer.

Amino Acid Analysis and Edman Sequencing Methods.

Samples for amino acid analysis were dissolved in 50% (v/v)methanol-water and subjected to hydrolysis and analysis, essentially asdescribed (Castillo et al., FEMS Lett. 255: 296-300, 2006). AutomatedEdman sequencing was performed on an Applied Biosystems cLC system.

Immunosuppression and Toxicity Tests.

Colutellin A, in matched studies with cyclosporin A, was examined forits ability to inhibit the activation of CD4±T cells for the productionof IL-2 (Umland et al., Am. J. Respir. Cell Mol. Biol. 20: 481-492,1999; Clark et al., J. Immunol. 162: 2546-2554, 1999). This test iscommonly taken as an indication of the potential that a compound may actas an immunosuppressant (Umland et al., Am. J. Respir. Cell Mol. Biol.20: 481-492, 1999; Clark et al., J. Immunol. 162: 2546-2554, 1999).Total spleen cells were isolated from C57/B 6 mice and then werepreactivated with ConA (1 ug/mL, Sigma-Aldrich, St. Louis, Mo.) for 2days. These cells were then treated for 4 hours with cross-linkedHamster anti-mouse CD3/CD28 (1 ug/mL, BD Bioscience) antibodies in theBruff's Medium containing 5% fetal bovine serum and 1× brefeldin A.After the activation, cells were fixed and analyzed for IL-2 productionin the activated CD4± T cells using APC-conjugated anti-mouse IL-2 andPE-conjugated anti-mouse CD4 antibodies by flow cytometry. In likemanner, in matched studies, both colutellin A and cyclosporin A wereexamined for their toxicity profiles. Blood was collected from healthyadult donors and peripheral blood mononuclear cells (PBMCs) werepurified using Histopaque 1077 (Sigma Aldrich, St. Louis, Mo.) accordingto the manufacturer's instructions. The cells, PBMCs, were cultured at1×10⁶ cells/ml in X-vivo 15 medium (Cambrex, Walkersville, Md.) withvarying concentrations of cyclosporin A, colutellin A or equivalentamounts of DMSO for 24 or 48 hours. Cells were washed twice with PBSfollowed by staining with Annexin V directly conjugated to PE or FITCand 7-AAD using the Annexin V Apoptosis Detection Kit I (BD Biosciences,San Jose, Calif.) as per the manufacturer's instructions. Cells werethen subjected to flow cytometry on a FACS Calibur equipped with an HTSloader (BD Biosciences, San Jose, Calif.). The staining allowed fordifferentiation among viable, necrotic and apoptotic cells. The studieswere repeated at least three times and the variation between the deadcells detected was recorded as a function of concentration over 24 and48 hour test periods.

Example 2 Isolation of Fungal Endophytes

This example describes the isolation of fungal endophytes. Each of theplants collected in the Costa Rican rainforest yielded a largecollection of endophytic fungi. The stem tissues of Pteromischum sp.,however, supported the growth of a number of fungal colonies that provedto be identical to each other and are collectively labeled CR-12. Noother plant in the same area yielded this fungus. The colonies werebrownish, round and discrete. Multiple sporodochia were locatedthroughout the surface of the fungal colonies. Each sporodochium hadseveral large setae possessing echinulated surfaces. The conidiophoreswere located close together and were macronematous to mononematous andirregularly branched. The conidia were aggregated in slimy masses. Eachconidium was cylindrical and slightly curved and rounded or slightlytapered at the ends. For critical point dried specimens the sporesaveraged 19.1×2.1 μm. Images obtained by ESEM were approximately thesame length, but averaged 2.68 μm in diameter (FIG. 1). Thus, it isapparent that the methods used to prepare specimens for regular SEMcaused some shrinkage of the spores. On the basis of these initialmorphological characteristics this dematiaceous hyphomycetous fungus wasidentified as Volutella sp. (Pers.) Sacc. having synonymy with othernamed genera including Sarcopodium, Psilonia, Tricholeconium, Thelephoraand Conoplea (Ellis, More Dematiaceous Hyphomycetes CommonwealthMycological Institute, KEW, England, pg 507, 1976).

Upon further review and molecular characterization studies thisdematiaceous hyphomycetous fungus was identified as Colletotrichum sp.instead of as Volutella sp. Further identification of CR-12 was doneusing an ITS-5.8S rDNA analysis followed by a BLAST search. Thesestudies indicated that the closest relatives (at the 98% level) of thisfungus are various isolates of Colletotrichum spp., includingColletotrichum graminicola, and Colletotrichum capsici (C. capsici).Since C. graminicola is a fungal species designated for isolates ofColletotrichum that are pathogenic on corn (Zea mays), CR-12 was notgiven this species designation. However, CR-12 is also geneticallyrelated to C. capsici. It turns out that C. capsici is also a pathogenicfungus. Some researchers prefer to place non-pathogenic C. capsici-likefungi as a form of C. dematium, which is usually considered as anon-pathogenic taxon with slightly narrower conidia than C. capsici(Sutton, The Coelomycetes. Kew, UK: CMI 1980). The conidial shape andsize of CR-12 were also consistent with the assessment that this isolatebe designated C. dematium.

The rDNA sequences of CR-12 were deposited in GenBank under GenBankaccession number EU330193 (as available on Jul. 17, 2008) which ishereby incorporated by reference in its entirety as of Nov. 6, 2008.Because of its ability to inhibit a number of pathogenic fungi, using asimple assay test, this endophytic Colletotrichum sp. was selected forfurther study of its extracellular bioactive components by methods wellknown to those of skill in the art and as described in the Examples 3and 4 below.

Example 3 Isolation and Characterization of Colletotrichum sp.Endophytes

This example describes the isolation and characterization of anendophytic Colletotrichum sp.

The isolate C-12 of an endophytic Colletotrichum sp. yielded about 0.45mg/l of colutellin A (eluting from the HPLC column at 24.6 minutes)which possessed antimycotic activity. The compound had a mass of 1127.70and a sole millimolar UV extinction at 210 nm with

=1,014. This absorptivity is associated with the peptide bond and sinceno absorption bands appeared at 280 nm the compound was presumed not topossess any aromatic amino acids (Silverstein et al., SpectrometricIdentification of Organic Compounds Wiley & Sons, N.Y. pg 419, 1991).The NMR—proton spectrum was characteristic of a peptide having aliphaticcarbons along with more down field resonances occurring with carbonatoms bearing protons with adjoining nitrogen and oxygen atoms (FIG. 2)(Silverstein et al., Spectrometric Identification of Organic CompoundsWiley & Sons, N.Y. pg 419, 1991). The compounds found in the broadsilica-gel/HPLC peak at 23.9 min were subjected to ESI-QqTOF and LTQ-FTmass spectrometry revealing both singly- and doubly-charged molecularions of mass 1081.7, 1095.7, 1111.7, and 1127.7, of which the 1095.7peak was the most prominent (FIG. 3). Sodium adducts of these molecules,all of which were 22 atomic mass units (amu) higher in mass (FIG. 3),also were observed, the intensities of which were substantially reducedby desalting. Mass differences of 14 and 16 amu among the ions suggestthat the compounds are related by the presence or absence of oxygen ormethylene groups, the latter of which would be a common variation amonglipopeptides with the lipid component being modified. These compoundswere not further examined. Quantitative amino acid analysis ofcolutellin A revealed the presence of Ile, Val, Ser, N-methyl-Val, andβ-amino-isobutryic acid in nominal molar rations of 3:2:1:1:1,respectively. Both Edman and mass spectrometric sequencing that revealedan N-terminal tetrapeptide sequence Val-Ile-Ser-Ile (SEQ ID NO: 1) and atripeptide sequence Ile-Pro-Val. Signal levels of the first four aminoacids were significant during Edman sequencing, thus the lack ofadditional sequence suggested the peptide was blocked, likely by nativecyclization. The cyclic nature of the peptides was supported by theadded observations that colutellin A reacted poorly with ninhydrin(faint pink spot upon heating) and that sodiated ions were observed byMicromass LCT TOF mass spectrometry which is common for cyclic peptides.

An examination of the Chapman Hall database revealed that there are anumber of known compounds with masses of 1127 including aureofungin A,halichrondrin C, norhalichrondrin A, deisobutrylolivomycin A, onchidin,partricin A, mepartricin B tetrocarin F and vacidin A. None of thesecompounds matches the complete description of the peptide made byColletotrichum sp. described herein. Furthermore, there were no directmatches in the database for the other observed masses of 1081.7, 1095.7,or 1111.7. Therefore, this novel compound was named—colutellin A afterits source fungal organism. Also, in contrast, cyclosporin A has a massof 1202.6 which is 75 mass units lower than colutellin. Furthermore,colutellin A is not a derivative of cyclosporin A in that it does notpossess such residues, among others such as alanine, and N-methylleucine. Thus, it appears that colutellin A represents a family ofrelated lipopeptides and one of which has a mass of 1127.7.

Example 4 Biological Activities of Colutellin A

This example describes the biological activities of colutellin A.

Because colutellin A had certain characteristics resembling those ofcyclosporin A (cyclic peptide with antifungal activity) all biologicalassays were conducted in matched studies. Both of the compounds possessstrong inhibitory activity against Botrytis cinerea and Sclerotiniasclerotiorum which remains stable up to 288 hours (Table 1). The resultwith S. sclerotiorum is in close agreement with that of Rodriguez et al.(J. Appl. Microbiol. 100: 575-86, 2006) who reported that cyclosporin Apossessed an MIC of 0.1 μg per disc. Harel et al. (Mol. Plant. Microbe.Interact. 6: 682-693, 2006) showed that calcineurin plays a major rolein both sclerotial development and pathogenesis of S. sclerotiorum. Thecalcineurin pathway maybe involved in the pathogenic potential of thismajor fungal pathogen (Steinbach et al., Nat. Rev. Microbiol. 6:418-430, 2007). It has been suggested that cyclosporin A may affect S.sclerotiorum by virtue of inhibiting the calcineurin pathway. This mayalso be true of colutellin A since they have comparable patterns ofantimycotic activity (Table 1). These findings support the possible useof Botrytis and Sclerotinia as a quick initial system for screeningorganisms for the production of immunosuppressants.

Both cyclosporin A and colutellin A possess a relatively narrow spectrumof antimycotic activity with some organisms such as Pythium ultimum andTrichoderma vi ride not being affected and others are quite sensitive(Table 1). Overall, it is also worth noting that although the test fungiare nicely matched relative to their sensitivities to the two compoundsexamined, the MIC values of cyclosporin A and colutellin A are, in somecases, more than 50 times different (Table 1). This suggests that thecompounds may have different molecular targets within some of the testorganisms.

TABLE 1 Minimum Inhibitory Concentrations (MICs) of Colutellin A andCyclosporin A on Common Fungal Pathogens Cyclosporin A Colutellin A MIC(μg/ml) (After h) MIC (μg/ml) (After h) Fungus tested 48 h 144 h 288 h48 h 144 h 288 h Pythium ultimum >100 >100 >100 >100 >100 >100Trichoderma virde >100 >100 >100 >100 >100 >100 Sclerotinia sclerotiorum0.07 0.1 0.1 3.6 10.8 32.4 Botrytis cinerea 0.07 0.1 0.1 3.6 10.8 10.8Fusarium solani >100 >100 >100 7.2 >100 >100 Rhizoctonia solani 1.2 10.810.8 >100 >100 >100 Aspergillus fumigatus 1.2 3.6 3.6 2.4 >100 >100Geotrichum canididum >100 >100 >100 3.6 >100 >100

Both colutellin A and cyclosporin A were examined for their ability toinhibit the IL-2 production by activated CD4± T cells. Generally, theinhibition of IL-2 production is directly related to the ability of acompound to act in a whole biological system as an immunosuppressant(Umland et al., Am. J. Respir. Cell Mol. Biol. 20: 481-492, 1999; andClark et al., J. Immunol. 162: 2546-2554, 1999).

Using all of the appropriate controls and various concentrations ofcolutellin A and cyclosporin A, IL-2 production for each compound wasplotted and then calculated in mouse spleen cells activated with ConA.The IC₅₀ for cyclosporin A was 61.8 nM and for colutellin A it was 167.3nM. The IC₅₀ of colutellin A is in the same range as cyclosporin Agiving an indication that colutellin A possesses immunosuppressiveproperties.

As a result of the enormous importance of cyclosporin A as the firstwidely used immunosuppressive compound, a search for other naturalproducts with similar activity was initiated. Although a large number ofnatural products have demonstrated immunosuppressant activity, moststudies have not included the corresponding cytotoxic activity of thereported compounds (Mann, Nat. Product Repts. 18: 417-430, 2001).Interestingly, the use of cyclosporin A in clinical settings has to becarefully monitored because of its cytotoxic activity. In this regard,its toxicity was compared with colutellin A in studies with humanperipheral blood mononuclear cells (PBMCs). Cyclosporin A exhibited ahigher level of cytotoxicity on human PBMCs than colutellin A or DMSOalone after 24 and 48 hours of culture (FIG. 4). Specifically, inrepeated tests, at concentrations at or above Bug/ml cyclosporin inducessignificant levels of both necrosis and apoptosis, whereas colutellin Adid not induce significant cell death above the DMSO controls at anyconcentration tested (FIG. 4).

Because of the immense importance of cyclosporin A to medicine, acomprehensive search was undertaken to find other organisms producingcyclosporin A and/or its derivatives. At least 28 natural cyclosporinshave been discovered being produced by 25 different fungal taxa (Lawenet al., Biochem. J. 300: 395-399, 1994; Jegorov et al., Phytochem. 38:403-407, 1995; Traber et al., J. Ind. Microbiol. &Biotech. 17: 397-401,1996). Some of these include: Acremonium luzulae, Aphanocladium sp.,Beauveria brongniarti, Chaunopycnis alba, Cylindrotrichum oligospermum,Cylindrocarpon lucidum, Fusarium oxysporum, Fusarium solani, Isariafelina, Neocosmospora africana, Paecilomyces spp., Stachybotryschartarum, and Tolypocladium spp. (Sakamoto et al., J. Antibiot. 46:1788-1798, 1993; Dreyfuss and Chapela., Biotech. 26: 49-80.1994; Jegorovet al., Phytochem. 38: 403-407, 1995; Traber et al., J. Ind. Microbiol.&Biotech. 17: 397-401, 1996). In addition, about 800 semisynthetic orsynthetic analogs have been produced and tested in vitro, but only a fewof them were worth testing in vivo (Rehacek, Folia Microbiol 40: 68-881995). Most of the immunosuppressant compounds isolated from nature arelipopeptides, cyclic peptides, or cyclic lipopeptides, but few have lowcytotoxicity accompanied with high immunosuppressive activity. This factmakes colutellin A a desirable drug, since it has little or no toxicityand significant immunosuppressive activity.

Example 5 Method of Suppressing an Immune Response

This example illustrates a representative method of suppressing orinhibiting an immune response, such as an immune response associatedwith an organ or tissue transplantation, an autoimmune disease or anon-autoimmune inflammatory disease.

According to the teachings herein, one can prevent, suppress, or inhibitan immune response by administering an endophytic Colletotrichum sp.isolate from a Pteromischum sp. plant, or a composition including one ormore Colletotrichum sp. compounds (such as a cyclic lipopeptide). Forexample, a subject who is in need of immunosuppression, such as asubject with an autoimmune disease, a non-autoimmune inflammatorydisease or an organ or tissue transplant recipient is selected. Atherapeutically effective amount of a Colletotrichum sp. isolate (suchas C-12 isolate) or compound (such as a compound including aColletotrichum sp. isolate with a molecular mass of 1081.7, 1095.7,1111.7, 1127.7 or a combination of two or more such compounds) isadministered to the subject to prevent, inhibit, or suppress the immuneresponse associated with the disease or the transplantation. Aneffective amount of the Colletotrichum sp. isolate or compound to beused will depend, at least, on the particular method of use, the subjectbeing treated, the severity of the infection, and the manner ofadministration of the therapeutic composition. For example, this can bethe amount of the Colletotrichum sp. isolate or compound necessary toprevent, inhibit, or suppress an immune response associated with thegiven condition. Ideally, a therapeutically effective amount is anamount sufficient to prevent, inhibit, or suppress the immune responsewithout causing a substantial cytotoxic effect on host cells. Thecompounds are prepared as described herein. In a specific example, theColletotrichum sp. isolate or compound (such as a Colletotrichum sp.compound including colutellin A) is administered to prevent, inhibit, orsuppress an immune response associated with an autoimmune disease,non-autoimmune disease or an organ or tissue transplantation. Forexample, the composition including endophytic colutellin A, B, C, D or acombination thereof suppresses the immune response by at least 10%, suchas at least 20%, at least 30%, at least 50%, at least 70%, at least 80%,at least 90% or at least 95%.

Exemplary autoimmune diseases affecting mammals include rheumatoidarthritis, juvenile oligoarthritis, collagen-induced arthritis,adjuvant-induced arthritis, Sjogren's syndrome, multiple sclerosis,experimental autoimmune encephalomyelitis, inflammatory bowel disease(e.g., Crohn's disease, ulcerative colitis), autoimmune gastric atrophy,pemphigus vulgaris, psoriasis, vitiligo, type 1 diabetes, non-obesediabetes, myasthenia gravis, Grave's disease, Hashimoto's thyroiditis,sclerosing cholangitis, sclerosing sialadenitis, systemic lupuserythematosis, autoimmune thrombocytopenia purpura, Goodpasture'ssyndrome, Addison's disease, systemic sclerosis, polymyositis,dermatomyositis, autoimmune hemolytic anemia, pernicious anemia, and thelike. Further, exemplary inflammatory diseases affecting mammals includerheumatoid arthritis, osteoarthritis, inflammatory lung disease(including chronic obstructive pulmonary lung disease), inflammatorybowl disease (including ulcerative colitis and Crohn's Disease),periodontal disease, polymyalgia rheumatica, atherosclerosis, systemiclupus erythematosus, systemic sclerosis, Sjogren's Syndrome, asthma,allergic rhinitis, and skin disorders (including dermatomyositis andpsoriasis) and the like. In particular examples, a therapeuticallyeffective amount of an endophytic Colletotrichum sp. isolate, suchisolate C-12, a compound (such as a cyclic lipopeptide (e.g., colutellinA)), or a salt or ester thereof is administered to suppress suppressingan immune response associated with at least one or combination thereofof the aforementioned diseases.

Example 6 Methods of Protecting a Plant from a Plant Pathogen

This example illustrates methods of protecting a plant from a plantpathogen including use of pesticidal compositions and compounds.

Based upon the teachings herein, one can protect a plant from a plantpathogen by use of at least one isolated strain of Colletotrichum sp.which is an endophyte of a Pteromischum sp. plant or compounds andcompositions including one of the disclosed endophytic Colletotrichumsp. compounds. For example, pesticidal compounds and compositionsincluding Colletotrichum sp. compounds with a molecular mass of 1081.7,1095.7, 1111.7 and 1127.7 or a combination of two or more such compoundsare administered to plants, or the associated soil, equipment,containers, machinery, surfaces and the like to protect plants fromplant pathogens. Plant pathogens can include, but are not limited to,Botrytis cinerea, Sclerotinia sclerotiorum, and Rhizoctonia solani. Foruse with a plant, the method involves applying an isolatedColletotrichum sp. strain or an extract or compound derived from theisolate either directly to the plant, or to soil adjacent to the plant.In some cases the treatment is made to seeds, e.g., in the format ofseed coats, soaks, or other such applications. In certain circumstances,the isolate (rather than an extract or compound) is applied to grow inassociation with the plant and produce the biologically active compoundscapable of protecting the plant against plant pathogen attack.

An effective amount of the isolate, extract, compound or composition canbe the amount required to control a pest. For example, an effectiveamount is the amount of the substance sufficient to control a pestthrough killing or stunting of the growth of the pest or protecting aplant from pest infestation. The pesticidal compositions can include anendophytic Colletotrichum sp. compound (such as a peptide) in asubstantially pure form or as an extract from a whole broth culture ofan endophytic Colletotrichum sp. strain in dry, concentrated, or liquidform and a suitable pesticidal carrier. The substance is present in thecomposition at a concentration of from about 0.001% to about 60% (w/w).

The pesticidal compositions can also include a deposition agent whichassists in preventing the composition from drifting from the target areaduring application (e.g., as it is sprayed from a plane), or from beingblown away from the plant once it has been deposited. The depositionagent in the compositions is preferably a proteinaceous material, whichhas the added benefit of being palatable to the insect. Any animal orvegetable protein is suitable for this purpose, in dry or in liquidform. Examples of useful sources of protein which can be convenientlyand economically added to the composition include, but are not limitedto, soy protein, potato protein, soy flour, potato flour, fish meal,bone meal, yeast extract, and blood meal. Alternative deposition agentsinclude modified cellulose (carboxymethylcellulose), botanicals (grainflours, ground plant parts), non-phyllosilites (talc, vermiculite,diatomaceous earth), natural clays (attapulgite, bentonite, kaolinite,montmorillonite), and synthetic clays (Laponite). When utilized, thedeposition agent is present in the pesticidal compositions disclosedabove in an amount of between about 0.4% w/w and about 50% w/w,preferably between about 1% w/w and about 20% w/w.

The pesticidal compositions can also include an antifreeze/humectantagent which suppresses the freeze point of the product and helpsminimize evaporation when sprayed and which maintains deposit texturemaking the product more efficacious and palatable. Examples ofantifreeze/humectant agents include, but are not limited to, ethyleneglycol, propylene glycol, dipropylene glycol, glycerol, butyleneglycols, pentylene glycols and hexylene glycols. When utilized, theantifreeze/humectant agent is present in the pesticidal compositions ofthe present disclosure in an amount of between about 0.5% w/w and about25% w/w, preferably between about 2% w/w and about 15% w/w.

In addition, a pesticidal composition can include a surfactant in anamount where it acts as an emulsifying, a wetting, or a dispersingagent. Examples of such surfactants are anionic surfactants such ascarboxylates, for example, a metal carboxylate of a long chain fattyacid; N-acylsarcosinates; mono or di-esters of phosphoric acid withfatty alcohol ethoxylates or salts of such esters; fatty alcoholsulphates such as sodium dodecyl sulphate, sodium octadecyl sulphate orsodium cetyl sulphate; ethoxylated fatty alcohol sulphates; ethoxylatedalkylphenol sulphates; lignin sulphonates; petroleum sulphonates; alkylaryl sulphonates such as alkyl-benzene sulphonates or loweralkylnaphthalene sulphonates, e.g., butyl naphthalene sulphonate; saltsor sulphonated naphthalene-formaldehyde condensates; salts ofsulphonated phenol-formaldehyde condensates; or more complex sulphonatessuch as amide sulphonates, e.g., the sulphonated condensation product ofoleic acid and N-methyl taurine or the dialkyl sulphosuccinates, e.g.,the sodium sulphonate or dioctyl succinate. Further examples of suchsurfactants are non-ionic surfactants such as condensation products offatty acid esters, fatty alcohols, fatty acid amides or fatty-alkyl- oralkenyl-substituted phenols with ethylene oxide, block copolymers ofethylene oxide and propylene oxide, acetylenic glycols such as2,4,7,9-tetraethyl-5-decyn-4,7-diol, or ethoxylated acetylenic glycols.Further examples of such surfactants are cationic surfactants such asaliphatic mono-, di-, or polyamine as acetates, naphthenates or oleates;oxygen-containing amines such as an amine oxide of polyoxyethylenealkylamine; amide-linked amines prepared by the condensation of acarboxylic acid with a di- or polyamine; or quaternary ammonium salts.When utilized, the surfactant is present in an amount of between about0.5% w/w and about 25% w/w, preferably between about 1% w/w and about 8%w/w.

The pesticidal compositions can also include an inert material,preservative, a feeding stimulant, an attractant, an encapsulatingpesticide, a binder, a dye, an ultraviolet light protectant, a buffer, aflow agent, or other component to facilitate product handling andapplication for particular target pests as described above.

The pesticidal compositions are applied in a dry or liquid form, e.g., asuspension, a solution, an emulsion, a dusting powder, a dispersiblegranule, a wettable powder, an emulsifiable concentrate, an aerosol orimpregnated granule, or a concentrate or primary composition whichrequires dilution with a suitable quantity of water or other diluentbefore application. The concentrations of each component in thecomposition will vary depending upon the nature of the particularcomposition, specifically, whether it is a concentrate or to be useddirectly. The composition can contain about 1% to about 98% of a solidor liquid inert carrier. In an example, the compositions areadministered at the labeled rate for commercial products, preferablyabout 0.01 pound to 5.0 pounds per acre when in dry form and at about0.01 pint to 25 pints per acre when in liquid form.

In one example, the pesticidal compositions are applied directly to aplant by, for example, spraying or dusting at the time when the pest hasbegun to appear on the plant or before the appearance of pests as aprotective measure. Alternatively, the pesticidal compositions areapplied by foliar, furrow, broadcast granule, “lay-by”, or soil drenchapplications. Additional applications include applying the compositionsdirectly to ponds, lakes, streams, rivers, still water, and other areassubject to infestation by pests of concern to public health by spraying,dusting, sprinkling, or the like. The spray or dust can convenientlycontain another pesticide.

The pesticidal compositions are applied to protect a number of differentplant types, including, but not limited to, cereals (wheat, barley, rye,oats, rice, sorghum and related crops), beets (sugar beet and fodderbeet), drupes, pomes and fruit (apples, pears, plums, peaches, almonds,cherries, strawberries, raspberries, and blackberries), leguminousplants (alfalfa, beans, lentils, peas, soybeans), oil plants (rape,mustard, poppy, olives, sunflowers, coconuts, castor oil plants, cocoabeans, groundnuts), cucumber plants (cucumber, marrows, melons), fiberplants (cotton, flax, hemp, jute), citrus fruit (oranges, lemons,grapefruit, mandarins), vegetables (spinach, lettuce, asparagus,cabbages and other brassicae, carrots, onions, tomatoes, potatoes),lauraceae (avocados, cinnamon, camphor), deciduous trees and conifers(linden-trees, yew-trees, oak-trees, alders, poplars, birch-trees, firs,larches, pines), or plants such as maize, turf plants, tobacco, nuts,coffee, sugar cane, tea, vines, hops, bananas and natural rubber plants,as well as ornamental plants and particularly plants which are grown fortheir flowers.

Deposit of Biological Material

If necessary, strains disclosed herein will be deposited underconditions that assure that access to the culture(s) will be availableduring the pendency of this patent application to one determined by theCommissioner of Patents and Trademarks to be entitled thereto under 37C.F.R. §1.14 and 35 U.S.C. §122. Each deposit will represent asubstantially pure culture of the deposited strain. However, it shouldbe understood that the availability of a deposit does not constitute alicense to practice the subject disclosure in derogation of patentrights granted by governmental action. All restriction on theavailability to the public of the material so deposited will beirrevocably removed upon the granting of a patent.

While this disclosure has been described with an emphasis uponparticular embodiments, it will be obvious to those of ordinary skill inthe art that variations of the particular embodiments may be used, andit is intended that the disclosure may be practiced otherwise than asspecifically described herein. Features, characteristics, compounds, orexamples described in conjunction with a particular aspect, embodiment,or example of the disclosure are to be understood to be applicable toany other aspect, embodiment, or example of the disclosure. Accordingly,this disclosure includes all modifications encompassed within the spiritand scope of the disclosure as defined by the following claims.

What is claimed is:
 1. A method of suppressing an immune response,comprising: administering to a subject in need of immunosuppression atherapeutically effective amount of a compound which is a peptideobtained from Colletotrichum dematium, wherein the peptide has amolecular mass of about 1127.7 Da and a tetrapeptide sequenceVal-Ile-Ser-Ile (SEQ ID NO:1) thereby suppressing the immune response.2. The method according to claim 1, wherein the compound has biologicalactivity against a fungal plant pathogen.
 3. The method according toclaim 2, wherein the fungal plant pathogen is at least one of Botrytiscinerea, Sclerotinia sclerotiorum, or Rhizoctonia solani.
 4. The methodof claim 1, further comprising first selecting a subject in need ofimmunosuppression.
 5. The method of claim 1, wherein the peptidecontains residues of Ile, Val, Ser, N-methyl-Val andbeta-aminoisobutryic acid in nominal molar ratios of 3:2:1:1:1,respectively.
 6. The method of claim 1, wherein the compound isadministered in the form of a composition which comprises Colletotrichumdematium and the compound.
 7. The method of claim 1, wherein thecompound is administered in the form of a crude extract ofColletotrichum dematium which comprises the compound.